Assemblies of concanavalin A onto carboxymethylcellulose

The immobilization of Concanavalin A, (Con A), onto flat surfaces formed by ultrathin films of carboxymethylcellulose, CMC, silicon wafers or spin-coated poly(methyl methacrylate), (PMMA), was studied by ellipsometry, contact angle measurements and atomic force microscopy (AFM). The formation of Con A monolayer was only observed onto CMC films. The adsorption constant of Con A onto CMC films was determined as being (2.1+/-0.2) x 10(6) L mol(-1). After assembling Con A onto CMC surfaces, these became more hydrophobic, indicating a molecular orientation of Con A hydrophilic residues to the polysaccharide and Con A hydrophobic residues to the air. The affinity of Con A for hydroxyl rich silicon surfaces or for more hydrophobic PMMA films was very weak, evidencing that nonspecific interactions play a marginal role. For comparison, the immobilization of Con A onto hybrid particles of PMMA/CMC was investigated by means of UV-spectrophotometry. Such particles carry CMC chains attached to the surface, as evidenced by mean zeta potential value of -40 mV. The adsorption constant determined for Con A onto PMMA/CMC particles was one order of magnitude smaller than that found for Con A onto CMC films. This finding indicates that the substrate geometry might influence the molecular arrangement of sugar residues on the surface, consequently affecting the sugar-Con A interaction (cluster effect).     

RAFT-mediated synthesis of cationic poly[(ar-vinylbenzyl)trimethylammonium chloride] brushes for quantitative DNA immobilization

The synthesis of cationic poly[(ar-vinylbenzyl)trimethylammonium chloride)] [poly(VBTAC)] brushes was achieved via reversible addition-fragmentation chain transfer (RAFT) polymerization and used for quantitative DNA immobilization. Initially, silicon surfaces were modified with RAFT chain transfer agent by utilizing an amide reaction involving a silicon wafer modified with allylamine and 4-cyanopentanoic acid dithiobenzoate (CPAD). Poly(VBTAC) brushes were then prepared via RAFT-mediated polymerization from the surface immobilized CPAD. Various characterization techniques including ellipsometry, X-ray photoelectron spectroscopy, grazing angle-Fourier transform infrared spectroscopy, atomic force microscopy and contact-angle goniometer were used to characterize the immobilization of CPAD on the silicon wafer and the subsequent polymer formation. The addition of free CPAD was required for the formation of well-defined polymer brushes, which subsequently resulted in the presence of free polymer chains in solution. The free polymer chains were isolated and used to estimate the molecular weights and polydispersity index of chains attached to the surface. Moreover, from atomic force microscopy and ellipsometry measurements, it was also determined that the density of immobilized DNA on the cationic poly(VBTAC) brushes can be quantitatively controlled by adjusting the solution concentration.     

Electrodeposited biotinylated polypyrrole as an immobilization method for impedimetric immunosensors

The potentialities of an electrodeposited biotinylated polypyrrole film as an immobilization matrix for the fabrication of impedimetric immunosensors are described. Biotinylated antibody (anti-human IgG), used as a model system, was attached to free biotin groups on the electrogenerated polypyrrole film using avidin as a coupling reagent. The resulting recognition interface consisted of a highly oriented monolayer immobilized onto the polymer surface. Cyclic voltammetry was used to characterize the polymer film. Additionally, scanning electron microscopy and atomic force microscopy were used to investigate the morphology of the immobilized material. This immobilization method allows the obtention of a highly reproducible and stable device. The resulting immunosensor has a linear dynamic range of 10-80 ng.ml/sup -1/ of antigen and a detection limit of 10 pg.ml/sup -1/. Furthermore, this immunosensor exhibited minor loss in response after two regeneration steps.     

Preparation and tribological studies of self-assembled triple-layer films

A self-assembled triple-layer film was grafted onto a silicon surface with a simple three-step method. Firstly, 3-glycidoxypropyltrimethoxysilane molecules were self-assembled on silicon surfaces, then coupled to 3-aminopropyltriethoxysilane through a surface ring-opening reaction, and finally octadecyltrichlorosilane (OTS) molecules were attached to the resultant alkoxysilane-terminated surface via Si-O-Si bonds. The structure and morphology of this triple-layer film were characterized with various techniques, such as contact angle measurement, ellipsometry, X-ray photoelectron spectroscopy, and atomic force microscopy (AFM). The influence of different surface chemical groups on surface adhesion properties was identified using the AFM force-volume technique. The micro- and macro-tribological properties of the triple-layer film were evaluated by friction force microscopy and a ball-on-plate tribometer. The triple-layer film shows good adhesive resistance and can greatly reduce the micro- and macro-friction force. Moreover, compared to self-assembled monolayer of OTS, this triple-layer film exhibited much better wear-resistance. This improvement was mainly ascribed to the network structure of a lateral cross-linked polysiloxane layer formed within the film which can enhance the stability of the film.     

Oriented immobilization of immunoglobulin G onto the cuvette surface of the resonant mirror biosensor through layer-by-layer assembly of multilayer films

A new method for oriented immobilization of immunoglobulin G (IgG) onto the cuvette surface of the resonant mirror biosensor through layer-by-layer (LBL) assembly of multilayer films composed of avidin/gold nanoparticles (GNp)/protein A/IgG was developed. First, avidin was added in the biotin cuvette, and then injected GNp, followed by the injection of protein A for oriented immobilization of IgG. The rinsing with PBS was applied at the end of each assembly deposition for dissociating the weak adsorption. Second, IgG was added in the protein A-coated cuvette, and regenerated by incubation with 0.1 M glycine–HCL buffer. Third, different concentrations of IgG were measured by repeating the second process. Film assembling and properties of the interaction between protein A and IgG were studied by resonant mirror biosensor and electrochemical measurements. Results confirmed that IgG was successfully oriented on the protein A-coated cuvette surface by LBL assembly of multilayer films. The interaction response was dose-dependent which showed a linear range of 0.1 – 1.6 g L^− 1 IgG, with a detection limit of 8.7 mg L^− 1 estimated at a signal-to-noise ratio of 3. Moreover, the assay for oriented immobilization of IgG exhibited a good reproducibility and a favorable reusability. This method can provide a promising platform for fabricating immunoassay and immunosensor systems, protein reactors or protein-modified substrates, and affinity probes.     

Preparation of biocatalytic nanofibres with high activity and stability via enzyme aggregate coating on polymer nanofibres

We have developed a unique approach for the fabrication of enzyme aggregate coatings on the surfaces of electrospun polymer nanofibres. This approach employs covalent attachment of seed enzymes onto nanofibres consisting of a mixture of polystyrene and poly(styrene-co-maleic anhydride), followed by a glutaraldehyde (GA) treatment that cross-links additional enzyme molecules and aggregates from the solution onto the covalently attached seed enzyme molecules. These cross-linked enzyme aggregates, covalently attached to the nanofibres via the linkers of seed enzyme molecules, are expected to improve the enzyme activity due to increased enzyme loading, and also the enzyme stability. To demonstrate the principle, we coated α-chymotrypsin (CT) on nanofibres electrospun from a mixture of polystyrene and poly(styrene-co-maleic anhydride). The initial activity of CT-aggregate-coated nanofibres was nine times higher than nanofibres with just a layer of covalently attached CT molecules. The enzyme stability of CT-aggregate-coated nanofibres was greatly improved with essentially no measurable loss of activity over a month of observation under rigorous shaking conditions. This new approach of enzyme coating on nanofibres, yielding high activity and stability, creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, and biosensors.     

Immobilization of biomolecules onto surfaces according to ultraviolet light diffraction patterns

We developed a method for immobilization of biomolecules onto thiol functionalized surfaces according to UV diffraction patterns. UV light-assisted molecular immobilization proceeds through the formation of free, reactive thiol groups that can bind covalently to thiol reactive surfaces. We demonstrate that, by shaping the pattern of the UV light used to induce molecular immobilization, one can control the pattern of immobilized molecules onto the surface. Using a single-aperture spatial mask, combined with the Fourier transforming property of a focusing lens, we show that submicrometer (0.7 μm) resolved patterns of immobilized prostate-specific antigen biomolecules can be created. If a dual-aperture spatial mask is used, the results differ from the expected Fourier transform pattern of the mask. It appears as a superposition of two diffraction patterns produced by the two apertures, with a fine structured interference pattern superimposed.     

Preparation and properties of gold nanoparticle-electrodeposited titanium substrates with Arg-Gly-Asp-Cys peptides

Titanium metal has good biocompatibility, superior mechanical properties and excellent corrosion resistance. Like most metals, however, it exhibits poor bioactive properties and fails to bond to bone tissue. To improve its bioactivity, bioactive molecules, such as peptides, can be grafted onto titanium surfaces. In order to do this, the first step may be to establish a stable and compatible linking layer on the titanium surface. In this study, we used electrochemical methods to deposit gold (Au) nanoparticles onto titanium substrates, to which we then grafted arginine-glycine-asparagine-cysteine (RGDC) peptides by thiolate covalent coupling. Properties of electrodeposited Au nanoparticles were evaluated using a variety of techniques, including microstructural, chemical and electrochemical measurements. The biological responses of the RGDC-grafted Ti substrates were evaluated using MG3 human osteoblast-like cells. The results of thin-film X-ray diffraction (TFXRD) and scanning electron microscopy (SEM) indicated the polycrystalline orientation of Au nanoparticles deposited on the titanium surfaces with high density and controllable particle size. The RGDC peptide could be covalently bonded to Au-deposited Ti substrates via Au-thiolate species, as expected. Cell morphology showed that, on RGDC-immobilized titanium with Au particles, MG63 cells attached and spread more rapidly than on Ti substrates either without peptide or with direct loading of the peptide. Immunostaining for focal adhesion kinase (FAK) demonstrated that RGDC enhanced cell attachment. The present method for the formation of Au nanoparticles may serve as an alternative route for bioactive molecule immobilization on Ti implants.     

Cross-sectional STEM observation of nanoparticle-attached silicon wafer: specimen prepared by focused ion-beam

A thermal hydrosilylation process could successfully immobilize 5-heptene-1-thiol-stabilized gold nanoparticles onto hydrogen-terminated silicon surfaces. In order to understand the immobilization structures, it is very important to observe the linkage between the nanoparticles and the substrate surface. For this purpose, a cross-sectional observation of gold nanoparticle-attached silicon substrate was carried out by using a high resolution scanning transmission electron microscopy (HR-STEM). The specimens were prepared by using a focused ion-beam (FIB) machine. According to the Ga ion-beam irradiation, many single-nano-sized nanoparticles were fused to grow up to larger particles and amorphous Si layers were generated.     

Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers

The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s --> pi* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high-density" probe surface than on the "high-efficiency" probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.     

Microstructured bioreactive surfaces: covalent immobilization of proteins on Au(1 1 1)/silicon via aminoreactive alkanethiolate self-assembled monolayers

Micrometer-scale patterns of a defined surface chemistry and structure were produced on both ultraflat Au(1 1 1) and on gold-coated monocrystalline silicon surfaces by a method combining microcontact printing, wet chemical etching and the replacement of etch-resist self-assembled monolayers (SAMs) by functionalized or reactive SAMs. Key steps in this methodology were characterized by X-ray photoelectron spectroscopy (XPS), ellipsometry and contact angle measurements. The covalent immobilization of (functional) biological systems on these surfaces was tested using an N-hydroxysuccinimide ester -functionalized disulphide (DSU), which covalently binds primary amines without the need for further activation steps. Atomic force microscope images of native collagen V single molecules immobilized on these patterned surfaces revealed both high spatial resolution and strong attachment to the monolayer/gold surface. Microcontact printing of DSU is shown to be feasible on specially prepared, ultraflat Au(1 1 1) surfaces providing a valuable tool for scanning probe experiments with biomolecules. The retention of enzymatic activity upon immobilization of protein was demonstrated for the case of horseradish peroxidase. The described approach can thus be used to confine biological activity to predetermined sites on microstructured gold/silicon devices – an important capability in biomedical and biomolecular research. © 1999 Kluwer Academic Publishers     

Collagen immobilization on polyethylene terephthalate surface after helium plasma treatment

An attractive alternative to add new functionalities such as biocompatibility due to the micro- and nano-scaled modification of polymer surfaces is offered by plasma processing. Many vital processes of tissue repair and growth following injuries depend on the rate of adsorption and self-assembling of the collagen molecules at the interfaces. Consequently, besides the amount of protein, it is necessary to investigate the form in which the collagen molecules are organizing on the polymer surface. In this study, direct current (DC) helium plasma treatment was used in order to obtain poly(ethylene terephthalate) (PET) films with different amounts of collagen and different shapes of aggregates formed from the collagen molecules. The immobilization of collagen on PET surface was confirmed by XPS measurements, an increase of the nitrogen content by increasing the plasma exposure time being recorded. The SEM and AFM measurements revealed the presence of grains and dendrites of collagen formed on the polymer surface. At 15 min plasma treatment time, the polymer surface after collagen immobilization has a homogenous topography. Usually, one can find fibrils, coil or dendrimers of collagen formed in buffer solutions and immobilized on different polymer surfaces. On the other hand, in this particular configuration, the combination of DC plasma and helium gas as a PET functionalization tool is an original one. As the collagen is not covalently immobilized on the surfaces, it may interact with the cell culture medium proteins, part of the collagen might being replaced by other serum proteins.     

Solid‐Supported Biomolecules on Modified Silica Surfaces—A Tool for Fast Physicochemical Characterization and High‐Throughput Screening

Biofunctionalization for a wide variety of applications can be achieved by coating silica surfaces with biomolecules such as lipids or proteins. However, specific surface optimization of the inorganic SiO₂ is necessary to achieve biocompatible surfaces. Surface shielded porous silica beads can be non‐covalently coated with a single lipid bilayer. The lipids retain their fluidity in this handy solid‐supported system, perfectly mimicking the soft‐surface properties of cellular membranes. A supramolecular architecture can also be used for functional immobilization of membrane proteins: An artificial cytosolic compartment can be created with the aid of polymers; coating by lipid membranes and integration of membrane proteins results in a solid‐supported biofunctional cellular surface. Another surface modification enables a direct immobilization of human serum albumin (HSA) molecules onto silica surfaces. The HSA on this otherwise passivated surface provides a convenient material for the investigation of unspecific protein binding of pharmaceuticals on a high‐throughput scale.     

Mimicking cell membrane-like structures on alkylated silicon surfaces by peptide amphiphiles

We present a new strategy for flexible attachment of peptide amphiphiles on functionalized silicon surfaces. This method involves the production of an alkylated surface on which a lipidated peptide can then be attached through hydrophobic interaction. We applied this to two derivatives of amphiphilic peptide molecules with the same amino acid sequence (A-A-A-A-G-G-G-E-R-G-D) but different in alkyl chain lengths (palmitic acid, undecanoic acid). The basis of this work was to develop substrates which are more biocompatible and bioactive. The ultra-thin peptide amphiphile films were characterized using electrical impedance spectroscopy (EIS), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (ATR-FTIR) spectroscopy. The results demonstrated that the length of the alkyl chain in the peptide amphiphile affects the packing and coverage of the peptides on the silicon surface.     

Controlling immunoglobulin G orientation on a protein-A terminated bilayer system

In this study, a bilayer system composed of N-[3-trimethoxysilyl propyl]-ethylene diamine (TEDA) and protein-A on silicon wafer was prepared by a simple two-step procedure. Self-assembly deposition of TEDA at optimal conditions resulted in the formation of homogeneous self-assembled monolayers (SAMs) ~ 2.3 nm thick with the surface roughness ~ 0.38 nm. The height value of protein-A overlayer was found to be ~ 3.5 nm, which is within experimental error of the diameter of a single protein-A (3 nm). Immunoglobulin G (IgG) molecules were then immobilized on the bilayer system by protein-A – IgG specific interactions. Using this very simple approach, the IgG layer was formed almost of a monomolecular layer for longer adsorption time (~ 100 min), and it was packed densely for adsorption time longer than 100 min, which resulted in the increase of the amount of IgG immobilized. The use of a bilayer system composed of TEDA and protein A on silicon wafer opens the door for a fundamental understanding of how protein A affects IgG orientation on the surface and also indicates a useful guide to designing surfaces for applications such as immunosensors and biochips.     

Immobilization of epidermal growth factor on titanium and stainless steel surfaces via dopamine treatment

Titanium and stainless steel were modified with dopamine for the immobilization of biomolecules, epidermal growth factor (EGF). First, the treatment of metal surfaces with a dopamine solution under different pH conditions was investigated. At higher pH, the dopamine solution turned brown and formed precipitates. Treatment of the metals with dopamine at pH 8.5 also resulted in the development of brown color at the surface of the metals. The hydrophobicity of the surfaces increased after treatment with dopamine, independently of pH. X-ray photoelectron spectroscopy revealed the formation of a significant amount of an organic layer on both surfaces at pH 8.5. According to ellipsometry measurements, the organic layer formed at pH 8.5 was about 1000 times as thick as that formed at pH 4.5. The amount of amino groups in the layer formed at pH 8.5 was also higher than that observed in the layer formed at pH 4.5. EGF molecules were immobilized onto the dopamine-treated surfaces via a coupling reaction using carbodiimide. A greater amount of EGF was immobilized on surfaces treated at pH 8.5 compared with pH 4.5. Significantly higher growth of rat fibroblast cells was observed on the two EGF-immobilized surfaces compared with non-immobilized surfaces in the presence of EGF. The present study demonstrated that metals can become bioactive via the surface immobilization of a growth factor and that the effect of the immobilized growth factor on metals was greater than that of soluble growth factor.     

Oriented immobilization of antibodies onto the gold surfaces via their native thiol groups

The general approach for site-oriented immobilization of antibodies onto gold supports is reported. The immobilization is carried out using the native sulfide groups of immunoglobulin (IgG). To liberate the thiol groups, the intact IgG was split into two half-IgG fragments without destruction of the binding site of the antibody. The immobilization of half-IgG fragments on the gold surface was carried out by simple adsorption. The antigen binding capacity of the half-IgG modified gold supports is similar to that of the gold surfaces with the traditionally linked antibodies and is much higher than for nonspecifically adsorbed intact IgGs. The immobilized antibodies, according to the proposed approach, maintain high antigen binding constants. The immobilization procedure provides orientation of IgG fragments in terms of the similar distance between the binding site of the antibody and the surface of the gold support, which does not cause the distribution of the apparent affinity constants. The high operational stability of half-IgG modified gold electrodes makes them applicable for analytical applications.     

Nanostructured medical device coatings based on self-assembled poly(lactic-co-glycolic acid) nanoparticles

Here we present a new method for providing nanostructured drug-loaded polymer films which enable control of film surface morphology and delivery of therapeutic agents. Silicon wafers were employed as models for implanted biomaterials and poly(lactic-co-glycolic acid) (PLGA) nanoparticles were assembled onto the silicon surface by electrostatic interaction. Monolayers of the PLGA particles were deposited onto the silicon surface upon incubation in an aqueous particle suspension. Particle density and surface coverage of the silicon wafers were varied by altering particle concentration, incubation time in nanoparticle suspension and ionic strength of the suspension. Dye loaded nanoparticles were prepared and assembled to silicon surface to form nanoparticle films. Fluorescence intensity measurements showed diffusion-controlled release of the dye over two weeks and atomic force microscopy (AFM) analysis revealed that these particles remained attached to the surface during the incubation time. This work suggests that coating implants with PLGA nanoparticles is a versatile technique which allows drug release from the implant surface and modulation of surface morphology.     

Synthesis of 16-Mercaptohexadecanoic acid capped gold nanoparticles and their immobilization on a substrate

Controlled assembly of nanoparticles on substrates is a promising path to develop miniaturized electronic and optical devices. Among the important issues to be addressed in this area include immobilization of the nanoparticles on substrates in order to ensure that the system is robust. In this work, 16-mercaptohexadecanoic acid (16-MHDA) capped gold nanoparticles with a narrow size distribution have been synthesized through a single phase synthesis method and subsequently immobilized on to silicon surface through covalent molecular assembly. Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) confirmed the absence of unreacted thiol in the synthesized gold nanoparticles. Presence of gold nanoparticles on Si surface after the immobilization process was confirmed through XPS. Cross-sectional high resolution transmission electron microscopy (HR-TEM) images provide direct evidence that the particles are indeed anchored to the silicon surface. The formation of uniform-sized and separated acid functionalized gold nanoparticles and their immobilization on to Si provide a basis for further nano-structuring.     

Controlled formation of dielectric chain aggregates on material surfaces

This paper investigated the formation of chain aggregates from fine particles suspended in gas stream onto material surfaces under the action of electric field. The results showed that the shape of aggregate formed on material surface was greatly influenced by the field intensity and the surface condition of materials. In a weak electric field without corona discharge, particles tended to form clustered aggregates on a metal plate with smooth surface, but on a metal mesh and a porous alumina substrate, to form chain aggregate. On the other hand, in a corona discharge field, these surfaces were coated uniformly. Consequently, for forming chain aggregates on material surface, an electric field without corona discharge and a rough surface are necessary conditions. On rough surface, chain aggregates of dielectric particles or conductive particles grew from the protrusions of the surface and could form a rough and porous layer. When the external electric field was removed, the chain aggregates remained long time due to the Van der Waals forces. After sintered at proper temperature, the chain aggregates became fiber-like. The results indicate that the formation of chain aggregate can be controlled by electrostatic force, and sintering can be used as a method for increasing their mechanical strength.